Fluorescence correlation spectroscopy (FCS) and related methods, in their implementation in a microscope assemblage, are suitable for the investigation of molecular mobility and interactions in microscopic measurement volumes smaller than one femtoliter (1 fl =10−15 liter) and at low concentrations of less than 1 μMol (μM). In typical FCS experiments with a stationary measurement volume and, in particular, in aqueous solution, the average number of fluorescent-labeled molecules present in the measurement volume is low (<1,000). Brownian motion of the molecules causes their number to fluctuate about that average, resulting in corresponding fluctuations in the fluorescence signal about an average. Autocorrelation analysis of one detection channel and/or cross-correlation analysis of more than one detection channel makes possible a quantitative determination of, for example, the concentrations and diffusion properties of several fluorescent-labeled species and their interactions with one another.
In structured samples in particular, for example in biological cells, additional and often dominant fluctuations (especially drift) can occur in the signal. The slow motions of cell structures are one example. These motions are overlaid on the diffusion-related molecular motions.
A further influence on the measurement result is exerted by fluorescent-labeled molecules that are transiently or permanently immobilized by binding onto cell structures. Under illumination, their fluorescence typically is irreversibly extinguished after times ranging from 100 msec to seconds. This is referred to as “photobleaching.”
Very slow fluorescent aggregates, moving slowly through the measurement volume as a singular event, may likewise be present. Combinations of the above-described influences on measurement results are conceivable.
When the correlation functions of the signals are calculated, these drifts and fluctuations often constitute dominant contributions that overlie the molecular-diffusion contributions of interest, and often make them impossible to evaluate. While analytical or numerical models permitting a quantitative evaluation do exist for the correlation function of signal fluctuations as a result of molecular diffusion, such is not (or very seldom) the case for the instances mentioned. In conventional FCS, therefore, slow fluctuations make evaluation difficult or impossible.
The existing art corresponds to the implementation of FCS experiments using the commercially available FCS units/correlators of ALV (ALV-Laser-Vertriebsgesellschaft mbH, Langen, Germany), ISS (ISS Inc., Champaign, Ill., USA), and Zeiss (Carl Zeiss Jena GmbH, Jena, Germany).